Method of identification and quantification of biological molecules and apparatus therefore

ABSTRACT

A method of detecting binding between first member or members of a binding pair and corresponding second member or members of the binding pair is disclosed. The method comprises interacting a solid support onto which the first member or members of the binding pair being immobilized and arrayed with the corresponding second member or members of the binding pair, the corresponding second member or members of the binding pair being directly or indirectly tagged with a heavy atom; and determining a spatial distribution of the heavy atom over a surface of the solid support, thereby detecting the binding between the first member or members of the binding pair and the corresponding second member or members of the binding pair.

FIELD OF THE INVENTION

The present invention relates to a method and apparatus for identifyingand quantifying molecules present in a sample, in particular by means ofirradiating appropriately tagged molecules with a particle beam, such asan electron beam, obtaining an image of the tags and carrying out imageanalysis. The present invention find uses and provides majorimprovements in the fields of genomics, proteomics, functionalproteomics, glycomics and cellomics.

BACKGROUND OF THE INVENTION

In the past, genes, proteins, carbohydrates and cells where mainlystudied at isolation, greatly limiting the ability to elucidate arealistic picture of the complex array of biochemical processes takingplace in living cells. Genomics, proteomics, glycomics and cellomicstechniques, which evolved in this sequence during the last decade, aimat analyzing biochemical processes, as complex as these may be, in amore integrated fashion, aiming at looking at all or substantially allof the biochemical changes, large scale changes, as well as minutechanges, that take place in living cells under various conditions.

Presently, genomics, proteomics, glycomics and cellomics rely on severaltechnologies that are insufficiently quantitative, insufficientlysensitive and are characterized by a relatively low signal-to-noise(S/N) ratio and as such fail to provide a complete insight of cellfunction. These include the nowadays routine technology of nucleic acidmicroarrays (e.g., DNA microarrays, also referred to in the art as DNAchips) for analyzing nucleic acid molecules, such as DNA and RNA; theemerging technology of protein (e.g., antigen or antibody) microarrays(also known as protein chips); the recently revived and improvedtechnology of two-dimensional polyacrylamide gel electrophoresis(2D-PAGE), both latter techniques serve for analyzing protein molecules;the recently introduced carbohydrate microarrays for the analysis ofcarbohydrate molecules and various cellomics assays for the analysis ofintegrated cells.

DNA microarrays or DNA chips comprise a plurality of DNA strands (probesor targets) immobilized on a surface of a substrate, where probes ortargets of known identity are located in known and hence addressablelocations over the surface of the substrate In a typical assay in whicha DNA microarray is used in the analysis of nucleic acids, singlestranded molecules (targets or probes, respectively), typicallyoligonucleotides or cDNA, tagged with fluorescent markers, areinteracted with the substrate, resulting in hybridization of targets andprobes according to the DNA parity rules. Following appropriate washes,the chip is scanned, typically with a laser-scanner, which excites thefluorescent tags (where present) and reads the emitted light. Dependingon the application, the pattern of fluorescence over the surface of thechip provides information on the sequence of the targets and/or theexpression level of a variety of genes.

The basic limitation associated with the use of nucleic acid microarraysis the ‘flood’ of poor quality data. Currently about 90% of the data isinsignificant. In most cases, weakly expressed genes that can be veryimportant in a biological pathway, are not detected. This limitationarises from the poor signal-to-noise ratio (S/N) and insufficientsensitivity of this technique. It further leads to poor reproducibility.It is difficult to quantify the result of an experiment. The results ofseemingly identical experiments also vary considerably. Furthermore, inmany cases, the genes of most importance produce a weak signal that isnot at all detected.

Hence, there is a great need to increase the sensitivity and dynamicrange of microarrays and reduce their inherent noise and backgroundlevels.

These challenges are possibly achievable by substantially miniaturizingthe microarrays. The miniaturization is important since it will providea possibility to imprint larger portions of the genome on the same array(perhaps even the entire human genome). An additional reason forminiaturization is the long period of time required for the targetmolecules to cover an array by diffusion. The smaller the array, theshorter this time is, in a quadratic manner. nevertheless, the presentlyemployed analyzing techniques, i.e., the use of fluorescent tags andlaser scanning, impose great limitations on further miniaturization,both with respect to spatial resolution and with respect to scanningtime.

An additional type of limitations of the presently employed microarraysarise from the bleaching of the fluorescent tags once analyzed. After anarray is scanned, it is bleached, meaning that the fluorescent tags emitless than the required intensity of light. This property significantlyreduces the ability of a user to repeat a measurement of a pre-measuredexperiment.

Mainly due to the above limitations, there is no standard by whichmicroarray experiments are performed and/or analyzed, leaving a toolarge room for personal know-how. In many cases, experiments executed indifferent laboratories cannot be repeated or even compared.

Hence, it is evident that there is a great need for a microarraydetection system whose quantification is limited only by the biology.Preferably this system will be based on single molecule detection. Thissystem should be free of the limitations associated withfluorescence-based readings and advantageously should have the followingproperties: (i) high sensitivity; (ii) high S/N ratio; (iii)compatibility with miniaturization of the microarray, and with smallersample sizes; (iv) it should not bleach, providing the opportunity torescan a sample or its regions of interest more than once; and (v) itshould be able to incorporate assisted hybridization processes (not onlydiffusion).

One objective of the present invention is to disclose a microarray,scanning method and system, capable of performing high throughputdetection on the level of a single molecule. This system is sensitiveand reproducible enough to set the industry standard.

One option that may be considered is the use of a Scanning ForceMicroscope in the analysis of microarrays. However, the inherently lowthroughput of this system prevents it from being practical. The presentinvention solves the above mentioned limitations by means of scanningelectron microscopy.

An additional limitation, complementary to quantifying genes inmicroarrays, is the quantitative study of proteomics (the study ofproteins). Proteins determine many biological processes and are veryimportant to drug discovery and many other applications.

One tool in proteomics is high resolution two-dimensional polyacrylamidegel electrophoresis (2D PAGE) and the image analysis thereofElectrophoresis is the migration of charged molecules in a solution, inresponse to an electric field. The rate of migration depends on thestrength of the field, on the charge, size and shape of the molecules,as well as on the parameters of the medium through which the moleculesare moving. 2D gel electrophoresis is a method to separate moleculesthat differ in any combination of size or charge. The solution issupported by a gel (agarose, polyacrylamide), which prevents undesiredmigration (convection, diffusion) and sieves the molecules, thuscontributing to their separation on the basis of their sizes. In thepresent application, this system is referred to as 2D PAGE. The scope isnot limited to any particular gel.

2D PAGE systems typically resolve about 1000 proteins according to theirisoelectric propertied through a pH gradient in one direction andthereafter according to their size, in the presence of SDS, in a seconddirection, perpendicular to the first. The abundance of proteins in acell is within a range from single to millions of molecules. There aremany proteins in the gel that are not resolved, partly due to the lackof sensitivity of the separation and partly due to the lack ofsensitivity of the detection.

There are two typical phases in protein analysis: (i) separation of theproteins, e.g., via 2D PAGE; and (ii) analyzing the types of theseparated proteins, typically by mass spectrometry.

What is clearly missing is an intermediate stage where the number ofproteins in each spot is counted. Preferably the counting method will beable to distinguish between the different proteins. Preferably it willrely on single molecule detection.

Currently there is no technology that provides a satisfactoryquantitative answer to the issue of how many of the separated proteinsexist in each spot. An additional question is how many types of proteinsexist in each spot. Thus, there is a need for a technology that countsthe number of proteins before they are inserted in the massspectrometer.

The above-mentioned limitations of genomics and proteomics technologiesare amplified in the emerging technology of protein microarrays.

Protein microarrays or protein chips comprise a plurality of proteins(probes or targets) immobilized on a surface of a substrate, whereprobes or targets of known identity are located in known and henceaddressable locations over the surface of the substrate. In a typicalassay in which a protein microarray is used in the analysis of proteins,protein molecules (targets or probes, respectively), typically antigensor antibodies, tagged with fluorescent markers, are interacted with thesubstrate, resulting in binding of targets and probes according to theiridentity. Following appropriate washes, the chip is scanned, typicallywith a laser-scanner, which excites the fluorescent tags (where present)and reads the emitted light. Depending on the application, the patternof fluorescence over the surface of the chip provides information on theidentity of the targets and/or the level of their expression. In manycases, comparative competition assays are performed, where a change inthe pattern of fluorescence is indicative of the identity of the targetsand/or the level of their expression.

Protein microarrays will go a long way towards elucidating aspects ofcellular functions that DNA chips cannot provide, since measuring mRNAlevels alone ignores issues which are of great influence on cellularfunction, such as, but not limited to, protein lifetime, protein posttransnational modifications, etc. Protein chips find uses in two majorfields: drug discovery and diagnostics. In drug discovery, processessuch as drug candidate discovery and candidate optimization can begreatly assisted should highly sensitive and reliable protein chips andanalysis methods were available. In diagnostics, determining titers ofviruses and other pathogens, presence, absence or level of cancer andother markers, antibody profiles, etc., could be greatly assisted shouldhighly sensitive and reliable protein chips and analysis methods wereavailable.

It is apparent that proteomic tools are essential to obtain informationthat is unavailable when performing analysis on the gene level.Expressed genes can be subjected to significant post-translationalregulation, and proteins undergo significant post-translationalmodifications (such as phosphorylation, acetylation, etc.) thatsignificantly affect their function at the cellular level. In manycases, no correlation exists between the level of a specific messengerRNA and the level/activity of its encoded protein, because, most of thecontrol of the expression of that protein takes place at the posttranslational phase. To this effect, see, for example, S. P. Gygi, etal., Mol. Cell. Biol. 19 (1999) 1720-1730).

Recently it was shown that it is possible to array different proteins,or protein ligands on a microscope slide to study a variety of proteinfunctions (Macbeath et al., Nature 289, 2000). The basic challenge isthat the proteins bound on surface retain their activity and/or theirantigenic epitopes. The proteins attached covalently to the slidesurface yet retained their ability to interact specifically with othermacromolecules, e.g., other proteins, or with small molecules insolution. In this study the proteins attached to the slides were probedwith fluorescently-labeled proteins. Screening for protein-proteininteractions, substrates of protein kinases and targets of smallmolecules was demonstrated.

It often occurs that the less highly expressed proteins are those thatare of most interest since their response to various physiologicalstimuli is the most interesting and informative. Unfortunately, it seemsthat the low abundance proteins, such as hormones, cytokines, smallG-proteins, DNA binding proteins etc., are not easily detected by thepresent proteomics techniques (S. P. Gygi, Proc. Natl. Acad. Sci. USA 97(2000) 9390-9395). Detection on the single or close to single moleculelevel usually requires painstaking techniques that require tedioussample preparation and imaging and are hard to apply to high throughputmethods.

Carbohydrate microarrays comprise a plurality of carbohydrates (probesor targets) immobilized on a surface of a substrate, where probes ortargets of known identity are located in known and hence addressablelocations over the surface of the substrate. In a typical assay in whicha carbohydrate microarray is used in the analysis of carbohydrates,molecules such as antibodies directly or indirectly tagged withfluorescent markers, are interacted with the substrate, resulting inbinding of targets and probes according to their identity. Followingappropriate washes, the chip is scanned, typically with a laser-scanner,which excites the fluorescent tags (where present) and reads the emittedlight. Depending on the application, the pattern of fluorescence overthe surface of the chip provides information on the identity of thetargets/probes and/or the level of their expression. The limitationsdescribed hereinabove with respect to nucleic acid and proteinmicroarrays clearly apply also to carbohydrate microarrays.

Cell microarrays comprise a plurality of cells immobilized on a surfaceof a substrate, which cells can be screened for various properties in aliving or fixated state. The limitations described hereinabove withrespect to nucleic acid and protein microarrays clearly apply also tocarbohydrate microarrays.

There is thus a widely recognized need for, and it would be highlyadvantageous to have, a technique for the implementation of genomics,proteomics glycomics and cellomics devoid of the above limitations. Inparticular, it would be highly advantageous to have a technique forimplementing genomics, proteomics glycomics or cellomics that reachessingle molecule detection levels, yields high signal-to-noise ratios,and demonstrates a broad dynamic range, while, at the same time,retaining simple sample preparation, readily applicable for highthroughput screening.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method foridentification and quantification of biological molecules in a samplethat overcomes the drawbacks of the existing methods

The term biological molecule includes any molecule with biologicalrelevance. This includes, but is not limited to: polysaccharides, smallchemical molecules such as lipids, peptides, hormones and othermessengers, ATP GTP etc., drugs, non proteinaceous antigens and anyhomo- (e.g., protein-protein as example) and hetero- (e.g.,drug-protein, DNA-RNA, DNA-protein, etc.) complexes, as well aschemically modifications and derivatisations whether naturally occurringor man made, of all these different molecules.

It is another object of the present invention to provide such a methodfor identification and quantification of biological molecules in asample that is based on single-molecule detection, has a highersensitivity and signal-to-noise ratio and broader dynamic range thanexisting methods.

It is a further object of the present invention to provide such a methodfor identification and quantification of biological molecules in asample that is carried out without the bleaching that characterizesfluorescence-based detection, thus enabling measuring the same sampleseveral times.

It is a further object of the present invention to provide such a methodfor identification and quantification of biological molecules in asample that is carried out with an Environmental Scanning ElectronMicroscope (ESEM), thus enabling the investigation of a sample at almostatmospheric pressure.

It is a further object of the present invention to provide such a methodfor identification and quantification of biological molecules in asample that is carried out with a Wafer Inspection Scanning ElectronMicroscope (WISEM), thus greatly reducing the cost of instrumentationrequired to implement the method.

It is still a further object of the present invention to provide such amethod for identification and quantification of biological molecules ina sample that is carried out on a miniaturized microarray and allows toimprint larger portions of the human genome, or even the entire humangenome, on the same array.

According to one aspect of the present invention there is provided amethod of detecting binding between first member or members of a bindingpair and corresponding second member or members of the binding pair, themethod comprising interacting a solid support onto which the firstmember or members of the binding pair being immobilized and arrayed withthe corresponding second member or members of the binding pair, thecorresponding second member or members of the binding pair beingdirectly or indirectly tagged with a heavy atom; and determining aspatial distribution of the heavy atom over a surface of the solidsupport, thereby detecting the binding between the first member ormembers of the binding pair and the corresponding second member ormembers of the binding pair. Preferably, determining the spatialdistribution of the heavy atom over the surface of the solid support isat a dynamic range of linearity of at least four orders-of-magnitude.Still preferably, determining the spatial distribution of the heavy atomover the surface of the solid support is at a sensitivity of detectionequals to or greater than 1 of 10 binding events, e.g., i of 5 bindingevents, most preferably, about 1 of 1 binding events. Yet preferably,determining the spatial distribution of the heavy atom over the surfaceof the solid support is at a signal-to-noise ratio greater than 20,preferably greater than 50, more preferably greater than 80.

According to another aspect of the present invention there is provided amethod of detecting binding between first member or members of a bindingpair and corresponding second member or members of the binding pair, themethod comprising interacting a solid support onto which the firstmember or members of the binding pair being immobilized and arrayed withthe corresponding second member or members of the binding pair; anddetermining a spatial distribution of the second member or members ofthe binding pair at a dynamic range of linearity of at least fourorders-of-magnitude. Preferably, the corresponding second member ormembers of the binding pair are directly or indirectly tagged with aheavy atom, whereas determining the spatial distribution of the secondmember or members of the binding pair is by determining a spatialdistribution of the heavy atom over the surface of the solid support.Still preferably, determining the spatial distribution of the heavy atomover the surface of the solid support is at a dynamic range of linearityof at least four orders-of-magnitude. Yet preferably, determining thespatial distribution of the second member or members of the binding pairover the surface of the solid support is at a sensitivity of detectionequals to or greater than I of 10 binding events, e.g., equals to orgreater than I of 5 binding events, optimally the sensitivity is about 1of 1 binding events. Still preferably, determining the spatialdistribution of the second member or members of the binding pair overthe surface of the solid support is at a signal-to-noise ratio greaterthan 20, preferably greater than 50, more preferably, greater than 80.

According to still another aspect of the present invention there isprovided a method of detecting binding between first member or membersof a binding pair and corresponding second member or members of thebinding pair, the method comprising interacting a solid support ontowhich the first member or members of the binding pair being immobilizedand arrayed with the corresponding second member or members of thebinding pair; and determining a spatial distribution of the secondmember or members of the binding pair at a sensitivity of detectionequals to or greater than 1 of 10 binding events, preferably, thesensitivity equals to or greater than 1 of 5 binding events, morepreferably, the sensitivity equals to about 1 of 1 binding events. In apreferred embodiment, the corresponding second member or members of thebinding pair are directly or indirectly tagged with a heavy atom,whereas determining the spatial distribution of the second member ormembers of the binding pair is by determining a spatial distribution ofthe heavy atom over the surface of the solid support. Preferably,determining the spatial distribution of the second member or members ofthe binding pair over the surface of the solid support is at a dynamicrange of linearity of at least four orders-of-magnitude. Stillpreferably, determining the spatial distribution of the second member ormembers of the binding pair over the surface of the solid support is ata signal-to-noise ratio greater than 20, preferably, greater than 50,more preferably, greater than 80.

According to yet another aspect of the present invention there isprovided a method of detecting binding between first member or membersof a binding pair and corresponding second member or members of thebinding pair, the method comprising interacting a solid support ontowhich the first member or members of the binding pair being immobilizedand arrayed with the corresponding second member or members of thebinding pair; and determining a spatial distribution of the secondmember or members of the binding pair at a signal-to-noise ratio greaterthan 20, preferably, greater than 50, more preferably, greater than 80.Preferably, the corresponding second member or members of the bindingpair are directly or indirectly tagged with a heavy atom, whereasdetermining the spatial distribution of the second member or members ofthe binding pair is by determining a spatial distribution of the heavyatom over the surface of the solid support. Still preferably,determining the spatial distribution of the second member or members ofthe binding pair over the surface of the solid support is at a dynamicrange of linearity of at least four orders-of-magnitude. Yet preferably,determining the spatial distribution of the second member or members ofthe binding pair over the surface of the solid support is at asensitivity of detection equals to or greater than 1 of 10 bindingevents, preferably, the sensitivity equals to or greater than 1 of 5binding events, most preferably, the sensitivity is about 1 of 1 bindingevents.

According to further features in preferred embodiments of the inventiondescribed below, the binding pair is selected from the group consistingof antigen-antibody, antibody-antigen, hapten-antibody, antibody-hapten,nucleic acid-complementary nucleic acid, nucleic acid-substantiallycomplementary nucleic acid, ligand-receptor, receptor-ligand,enzyme-substrate, substrate-enzyme, enzyme-inhibitor andinhibitor-enzyme.

According to still further features in the described preferredembodiments determining the spatial distribution of the heavy atom overthe surface of the solid support is by particle scattering. Preferably,determining the spatial distribution of the heavy atom over the surfaceof the solid support is by electron scattering.

According to still further features in the described preferredembodiments the corresponding second member or members of the bindingpair is indirectly tagged with a heavy atom.

According to still further features in the described preferredembodiments the heavy atom is selected from the group consisting ofgold, silver and iron.

According to an additional aspect of the present invention there isprovided a method of identifying and/or quantifying at least onebiological molecule in a sample, the method comprising contacting thesample with a microarray presenting an addressable array ofmacromolecules of known identities under conditions so as to allowbinding between the at least one biological molecule and themacromolecules of known identities; detecting a spatial distribution ofthe at least one biological molecule over a surface of the microarray ata dynamic range of linearity of at least four orders-of-magnitude,thereby identifying and/or quantifying the least one biological moleculein the sample. Preferably, detecting the spatial distribution of the atleast one biological molecule over the surface of the microarray is at asensitivity equals to or greater than 1 of 10 binding events. Stillpreferably, detecting the spatial distribution of the at least onebiological molecule over the surface of the microarray is at asignal-to-noise ratio greater than 20. Yet preferably, detecting thespatial distribution of the at least one biological molecule over thesurface of the microarray is by directly or indirectly tagging the atleast one biological molecule with at least one heavy atom and obtaininga particle scattering image of a spatial distribution of the at leastone heavy atom.

According to still an additional aspect of the present invention thereis provided a method of identifying and/or quantifying at least onebiological molecule in a sample, the method comprising contacting thesample with a microarray presenting an addressable array ofmacromolecules of known identities under conditions so as to allowbinding between the at least one biological molecule and themacromolecules of known identities; and detecting a spatial distributionof the at least one biological molecule over a surface of the microarrayat a sensitivity equals to or greater than 1 of 10 binding events,thereby identifying and/or quantifying the least one biological moleculein the sample. Preferably, detecting the spatial distribution of the atleast one biological molecule over the surface of the microarray is at adynamic range of linearity of at least four orders-of-magnitude. Stillpreferably, detecting the spatial distribution of the at least onebiological molecule over the surface of the microarray is at asignal-to-noise ratio greater than 20. Yet preferably, detecting thespatial distribution of the at least one biological molecule over thesurface of the microarray is by directly or indirectly tagging the atleast one biological molecule with at least one heavy atom and obtaininga particle scattering image of a spatial distribution of the at leastone heavy atom.

According to yet an additional aspect of the present invention there isprovided a method of identifying and/or quantifying at least onebiological molecule in a sample, the method comprising contacting thesample with a microarray presenting an addressable array ofmacromolecules of known identities under conditions so as to allowbinding between the at least one biological molecule and themacromolecules of known identities; and detecting a spatial distributionof the at least one biological molecule over a surface of the microarrayat a signal-to-noise ratio greater than 20, thereby identifying and/orquantifying the least one biological molecule in the sample. Preferably,detecting the spatial distribution of the at least one biologicalmolecule over the surface of the microarray is at a dynamic range oflinearity of at least four orders-of-magnitude. Still preferably,detecting the spatial distribution of the at least one biologicalmolecule over the surface of the microarray is at a sensitivity equalsto or greater than 1 of 10 binding events. Yet preferably, detecting thespatial distribution of the at least one biological molecule over thesurface of the microarray is by directly or indirectly tagging the atleast one biological molecule with at least one heavy atom and obtaininga particle scattering image of a spatial distribution of the at leastone heavy atom.

According to still an additional aspect of the present invention thereis provided a method of identifying and/or quantifying at least onebiological molecule in a sample, the method comprising contacting thesample with a microarray presenting an addressable array ofmacromolecules of known identities under conditions so as to allowbinding between the at least one biological molecule and themacromolecules of known identities; and detecting a spatial distributionof the at least one biological molecule over a surface of the microarrayby directly or indirectly tagging the at least one biological moleculewith at least one heavy atom and obtaining a particle scattering imageof a spatial distribution of the at least one heavy atom, therebyidentifying and/or quantifying the least one biological molecule in thesample. Preferably, detecting the spatial distribution of the at leastone biological molecule over the surface of the microarray is at adynamic range of linearity of at least four orders-of-magnitude. Stillpreferably, detecting the spatial distribution of the at least onebiological molecule over the surface of the microarray is at asensitivity equals to or greater than 1 of 10 binding events. Yetpreferably, detecting the spatial distribution of the at least onebiological molecule over the surface of the microarray is at asignal-to-noise ratio greater than 20.

According to further features in preferred embodiments of the inventiondescribed below, the at least one biological molecule is selected fromthe group consisting of a protein, a glycoprotein, a nucleic acid and acarbohydrate.

According to still further features in the described preferredembodiments the macromolecules of known identities are selected from thegroup consisting of proteins, glycoproteins, nucleic acids andcarbohydrates.

According to another aspect of the present invention there is provided amethod of identifying and/or quantifying at least one biologicalmolecule in a sample, the method comprising attaching biologicalmolecules present in the sample to a solid support; contacting the solidsupport with at least one macromolecule of a known identity underconditions so as to allow binding between the at least one biologicalmolecule and the at least one macromolecule of known identity; anddetecting a level of binding between the at least one biologicalmolecule and the at least one macromolecule of known identity at adynamic range of linearity of at least four orders-of-magnitude, therebyidentifying and/or quantifying the least one biological molecule in thesample. Preferably, detecting the level of binding between the at leastone biological molecule and the at least one macromolecule of knownidentity is at a sensitivity equals to or greater than 1 of 10 bindingevents. Still preferably, detecting a level of binding between the atleast one biological molecule and the at least one macromolecule ofknown identity is at a signal-to-noise ratio greater than 20. Yetpreferably, detecting a level of binding between the at least onebiological molecule and the at least one macromolecule of known identityis by directly or indirectly tagging the at least one macromolecule ofknown identity with at least one heavy atom and obtaining a particlescattering image of a spatial distribution of the at least one heavyatom.

According to still another aspect of the present invention there isprovided a method of identifying and/or quantifying at least onebiological molecule in a sample, the method comprising attachingbiological molecules present in the sample to a solid support;contacting the solid support with at least one macromolecule of a knownidentity under conditions so as to allow binding between the at leastone biological molecule and the at least one macromolecule of knownidentity; and detecting a level of binding between the at least onebiological molecule and the at least one macromolecule of known identityat a sensitivity equals to or greater than 1 of 10 binding events,thereby identifying and/or quantifying the least one biological moleculein the sample. Preferably, detecting the level of binding between the atleast one biological molecule and the at least one macromolecule ofknown identity is at a dynamic range of linearity of at least fourorders-of-magnitude. Still preferably, detecting a level of bindingbetween the at least one biological molecule and the at least onemacromolecule of known identity is at a signal-to-noise ratio greaterthan 20. Yet preferably, detecting a level of binding between the atleast one biological molecule and the at least one macromolecule ofknown identity is by directly or indirectly tagging the at least onemacromolecule of known identity with at least one heavy atom andobtaining a particle scattering image of a spatial distribution of theat least one heavy atom.

According to a further aspect of the present invention there is provideda method of identifying and/or quantifying at least one biologicalmolecule in a sample, the method comprising attaching biologicalmolecules present in the sample to a solid support; contacting the solidsupport with at least one macromolecule of a known identity underconditions so as to allow binding between the at least one biologicalmolecule and the at least one macromolecule of known identity; anddetecting a level of binding between the at least one biologicalmolecule and the at least one macromolecule of known identity at asignal-to-noise ratio greater than 20, thereby identifying and/orquantifying the least one biological molecule in the sample. Preferably,detecting the level of binding between the at least one biologicalmolecule and the at least one macromolecule of known identity is at adynamic range of linearity of at least four orders-of-magnitude. Stillpreferably, detecting a level of binding between the at least onebiological molecule and the at least one macromolecule of known identityis at a sensitivity greater than or equals to 1 of 10 binding events.Yet preferably, detecting a level of binding between the at least onebiological molecule and the at least one macromolecule of known identityis by directly or indirectly tagging the at least one macromolecule ofknown identity with at least one heavy atom and obtaining a particlescattering image of a spatial distribution of the at least one heavyatom.

According to still a further aspect of the present invention there isprovided a method of identifying and/or quantifying at least onebiological molecule in a sample, the method comprising attachingbiological molecules present in the sample to a solid support;contacting the solid support with at least one macromolecule of a knownidentity under conditions so as to allow binding between the at leastone biological molecule and the at least one macromolecule of knownidentity; and detecting a level of binding between the at least onebiological molecule and the at least one macromolecule of known identityby directly or indirectly tagging the at least one macromolecule ofknown identity with at least one heavy atom and obtaining a particlescattering image of a spatial distribution of the at least one heavyatom, thereby identifying and/or quantifying the least one biologicalmolecule in the sample. Preferably, detecting the level of bindingbetween the at least one biological molecule and the at least onemacromolecule of known identity is at a dynamic range of linearity of atleast four orders-of-magnitude. Still preferably, detecting a level ofbinding between the at least one biological molecule and the at leastone macromolecule of known identity is at a sensitivity greater than orequals to 1 of 10 binding events. Yet preferably, detecting a level ofbinding between the at least one biological molecule and the at leastone macromolecule of known identity is at a signal-to-noise ratiogreater than 20.

According to further features in preferred embodiments of the inventiondescribed below, the at least one biological molecule is selected fromthe group consisting of a protein, a glycoprotein, a nucleic acid and acarbohydrate.

According to still further features in the described preferredembodiments the at least one macromolecule of known identity is selectedfrom the group consisting of a protein, a glycoprotein, a nucleic acidand a carbohydrate.

According to another aspect of the present invention there is provided amethod of identifying and/or quantifying biological molecules in apreparate, the method comprising localizing and tagging the biologicalmolecules in the preparate; preparing the preparate for vacuum; loadingthe preparate into the specimen chamber of an electron beam device;irradiating the preparate with an electron beam, thus obtaining an imageof the tags; and analyzing the image to quantity the biologicalmolecules by image analysis software.

According to another aspect of the present invention there is provided amethod of identifying and/or quantifying biological molecules in apreparate, the method comprising localizing and tagging the biologicalmolecules in the preparate; loading the preparate into the specimenchamber of an electron beam device; irradiating the preparate with anelectron beam, thus obtaining an image of the tags; and analyzing theimage to quantify the biological molecules by image analysis software.

According to still another aspect of the present invention there isprovided a method of identifying and/or quantifying biological moleculesin a preparate, the method comprising localizing the biologicalmolecules in the preparate; loading the preparate into the specimenchamber of an electron beam device; irradiating the preparate with anelectron beam, thus obtaining an image representing the biologicalmolecules; and analyzing the image to quantify the biological moleculesby image analysis software.

According to still another aspect of the present invention there isprovided an apparatus for inspection of a preparate of biologicalmolecules comprising an electron source to provide an electron beam; acharged particle beam column to deliver and scan an electron beam fromthe electron source on the surface of the preparate; a vacuum systemincluding a first and a second chamber in each of which pressurizationcan be performed independently to permit loading or unloading of a firstpreparate in one chamber while simultaneously inspecting a secondpreparate; at least one electron detector; means for measuring X-rayspectrum; a continuously moving x-y stage disposed to receive thepreparate and to provide at least one degree of motion to the preparatewhile the preparate is being scanned; and means for carrying out imageanalysis of the molecules on the preparate.

The biological molecules may be polynucleotides, e.g. DNA, cDNA, RNA,clusters, or proteins such as antigens, antibodies. The term biologicalmolecules also refers but is not limited to: polysaccharides, smallchemical molecules such as lipids, peptides, hormones and othermessengers, ATP antibodies, GTP, etc., drugs, non proteinaceous antigensand any homo-(protein-protein as example) and hetero- (drug-protein,DNA-RNA, DNA-protein etc.) complexes as well as chemically modificationsand derivatisations whether naturally occurring or not of all thesedifferent molecules. The preparate for the polynucleotides may be amicroarray, e.g., a DNA chip, and for the proteins may be a 2D PAGE, aprotein chip, e.g., an antigen or antibody chip, cell chip, cellpreparate, and the like.

The localization of the biological molecules may be carried out beforeor after tagging, depending on the type of the biological molecule andof the technique used.

When the biological molecule in the preparate is a polynucleotide, thelocalization may be carried out, for example, by hybridization, eitherto a polynucleotide of known sequence (probe) when the polynucleotideimmobilized in the microarray preparate is of unknown sequence (target),or to a polynucleotide of unknown sequence (target) when thepolynucleotide immobilized in the microarray preparate is of a knownsequence (probe). The same is true for protein microarrays, with respectto either antigens and/or antibodies, each of which can serve as atarget or probe, and in any case can be immobilized to the microarray orbe interacted therewith.

When the biological molecule in the preparate is a protein, thelocalization may be carried out, for example, by separating themolecules by one- or two-dimensional electrophoresis, or by attachingthe molecules to a blot membrane. When the preparate is a 2D PAGE orproteins extracted therefrom, the separation in the gel may bepreferably performed on-line under the scanning electron beam.Identification of the proteins can be done by mass spectrometry.

The localization in space may further consist of localizing themolecules by their affiliation to specific biological cells.

Tagging of the biological molecules such as DNA, RNA and proteins, maybe carried out with heavy metals such as silver or gold, for exampleusing colloidal gold or gold clusters, or doping with metal-enrichedorganic compounds, wherein the metal is, for example, Fe. The heavymetal colloids (e.g., gold), preferably of diameter range of 1-200 nm,more preferably, less than 20 nm, create a high intensity back scatteredelectron signal and, therefore, high image contrast. In one embodiment,there is one tag per target molecule. More specifically the tagging maybe carried out with biotin followed by gold tagged avidin.

Tagging may also be made with electro-luminescent molecules whereby theelectron beam creates a light signal that is detected. Tagging may alsobe done with more than one type of tags to make a distinction betweentwo preparates.

According to one embodiment multi-labeling or Multi-tagging is achieved.This is achieved, for example, by using gold colloids of a plurality ofsizes. According to another embodiment, multi-labeling is achieved byusing a combination of gold colloids and fluorescent labels. Accordingto a yet another embodiment, the multi-labeling is achieved by using aplurality of metals that are read by the X-RAY reading apparatus of theSEM, such as Energy Depressive Spectrum and so forth.

According to one embodiment, the DNA molecules are not tagged and theSEM is sensitive enough to detect density differences between hybridizedand non-hybridized regions. Direct detection with no tagging enables theidentification of an additional variety of substances such as viralparticles.

The preparates are prepared for vacuum by known standard methods thatinclude drying, fixation and coating with a conductive layer such ascarbon, to prevent charge accumulation., protection with a membrane andfreezing to prevent out-gassing.

The preparates are examined in a particles beam device, preferably, anelectron beam device, namely an electron microscope such as a scanningelectron microscope (SEM). Presently, most preferably, the preparatesare scanned and are analyzed using a wafer inspection SEM (WISEM)typically used in the microelectronics industry. The irradiation of thepreparate is carried out in such a way as to form sufficient contrast ofthe electrons that are back scattered from the tags in comparison withthose that are emitted/scattered from the background.

In another embodiment, the SEM system is an environmental scanningelectron microscope (ESEM) that works at almost atmospheric pressure,thus minimizing the need to prepare the preparate for vacuum.

According to a yet another embodiment, the SEM system that allows theproteins to remain in their native wet state and still imaged. Thisembodiment utilizes a device and method that uses membrane partition. Tothis end, see U.S. Provisional Patent Application No. 60/250,879, whichis incorporated herein by reference.

The image analysis may comprise any one of: performing edge detectionalgorithm to identify the colloids in each region-of-interest (ROI) andcounting the colloids; counting fluorescence signals; and identifyingX-ray spectrum of each particle for identification by comparison to areference spectrum. The invention further relates to an apparatus forinspection of a preparate of biological molecules according to the abovemethod, the apparatus comprising an electron source to provide anelectron beam; a charged particle beam column to deliver and scan anelectron beam from the electron source on the surface of the preparate;a vacuum system including a first and a second chamber in each of whichpressurization can be performed independently to permit loading orunloading of a first preparate in one chamber while simultaneouslyinspecting a second preparate; at least one electron detector; means formeasuring X-ray spectrum; a continuously moving x-y stage disposed toreceive the preparate and to provide at least one degree of motion tothe preparate while the preparate is being scanned by the electron beam;and means for carrying out image analysis of the molecules on thepreparate.

In one preferred embodiment, the charged particle beam column of is amicrocolumn.

In a further embodiment, the invention provides a method for theinspection of biological molecules on a preparate using an electronbeam, the method comprising localizing the biological molecules in spaceand tagging them with markers; preparing the preparate for vacuum;taking out the preparate to be analyzed from the preparate cassette;pre-aligning preparate and read preparate number; reading a recipe thatcontains the information to be detected; loading the preparate on X-Y-Tstage (T means tilt) of an electron beam device; aligning the preparate;moving XYT stage to analysis position; positioning the electron beam onthe substrate accurately by measuring the position of the substrate;scanning the preparate at low resolution to create a preparate map,while enhancing contrast; determining the regions-of-interest (ROI)spots on the map that should be scanned in a high resolution; scanningthe ROIs with the electron beam as the substrate is continuously movingwith at least one degree of motion in an x-y plane; detecting electronsemanating from the substrate as a result of previous step and forming animage; enhancing the image contrast; storing both modified and bareimage; analyzing the ROIs; and displaying the results.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing a technique for theimplementation of genomics, proteomics, glycomics and cellomics thatreaches the highest sensitivity of ultimately single molecule detection,yields high signal-to-noise ratios, and demonstrates a broad dynamicrange, while, at the same time, retaining simple preparate preparation,readily applicable for high throughput screening.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIG. 1 is a flow-chart illustrating steps of a method according to theteachings of the present invention.

FIG. 2 is a general diagram showing a longitudinal cross-section of anSEM used as a preparate analysis apparatus according to the presentinvention.

FIG. 3 is a generalized diagram showing a cross-section of a scanningelectron microscope according to an aspect of the present invention.

FIG. 4 shows an apparatus that combines an ESEM with a SEM according tothe present invention.

FIG. 5 presents another embodiment of the present invention, whereby thedetection is made by exciting light photons (electro-luminescence).

FIG. 6 a SEM device having a microcolumns array for use in the method ofthe present invention.

FIG. 7 is a gel chamber for use in the method of the present invention.

FIG. 8 demonstrates tagged proteins immobilized on a surface accordingto the present invention.

FIG. 9 is an image showing gold conjugate proteins as imaged in SEMwithout image processing according to the present invention.

FIG. 10 is a bar graph and a table demonstrating signal (S) tobackground (B) ratios (defined as (S-B)/B) for STP20, STP40 and Cy3-STPprobes in the BSA-biotin—gold/Cy3-streptavidin detection system. Onlylast five dilutions of BSA-biotin are shown.

FIG. 11 is a blow-up of FIG. 10, where only last three dilutions ofBSA-biotin are shown.

FIG. 12 is a bar graph demonstrating signal (S) to background (B) ratios(defined as (S-B)/B) for STP20 and Cy3-STP probes. The data for Cy3-STPprobes was obtained by averaging over results of 4 independent slides.

FIG. 13 is a graph demonstrating the estimated detection abilitiespresented as the number of biotinylated BSA molecules detected withSTP20 probe versus the total number of biotinylated BSA moleculespresent in a spot on the slide. The number of biotinylated BSA moleculesconjugated to the slide is approximated by calculating the number ofmolecules being able to attach to the surface. As shown in thecalculations below, this is at most 10% of the total molecules containedin a 10 ml drop that was spotted on the slide. This is the upper limitto this number, since most likely less then 10% of the moleculescontained in the drop indeed conjugated to the glass surface. The numberof molecules detected is given by the number of gold colloids detected.This number was calculated by extrapolating from the average number ofgold colloids counted in a single SEM frame (see, FIG. 14) to the areaof the whole drop. The dashed line represents a linear fit. From theslope of the fit and the efficiency of attachment of biotinylated BSA tothe slide it arises that the detection ability if of between 1:1 and1:4, hence, the upper limit of detection (sensitivity) was reached, overan unprecedented dynamic range.

FIG. 14 is a backscattered electrons image demonstrating single moleculedetection using 20 nm gold colloids according to the present invention.The high quality is achieved using backscattered electrons in accordancewith the teachings of the present invention. The number of gold colloidscan easily and accurately be quantified manually or via using a simpleimage analysis software.

FIG. 15 is a graph demonstrating signal (S) to background (noise, B)ratios (defined as (S-B)/B) for STP40 probe in theBSA-hapten—biotinylated antibody—gold streptavidin detection system ofthe present invention employing different dilutions of the biotinylatedantibody.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to a method and apparatus useful in theimplementation of genomics, proteomics, glycomics and cellomics. Inparticular, the method and apparatus of the present invention allowshighest sensitivity of ultimately single molecule detection, yields highsignal-to-noise ratios, and demonstrates a broad dynamic range, while,at the same time, retaining simple sample preparation, readilyapplicable for high throughput screening.

According to one aspect of the present invention there is provided amethod of detecting binding between first member or members of a bindingpair and corresponding second member or members of the binding pair. Themethod according to this aspect of the invention is implemented byinteracting a solid support onto which the first member or members ofthe binding pair are immobilized and arrayed with the correspondingsecond member or members of the binding pair. The corresponding secondmember or members of the binding pair are directly or indirectly taggedwith a heavy atom. Thereafter, a spatial distribution of the heavy atomover a surface of the solid support is determined, thereby bindingbetween the first member or members of the binding pair and thecorresponding second member or members of the binding pair is detected.

According to another aspect of the present invention there is provided amethod of detecting binding between first member or members of a bindingpair and corresponding second member or members of the binding pair. Themethod according to this aspect of the invention is implemented byinteracting a solid support onto which the first member or members ofthe binding pair is immobilized and arrayed with the correspondingsecond member or members of the binding pair. Thereafter, the spatialdistribution of the second member or members of the binding pair isdetermined at a dynamic range of linearity of at least four, preferablyat least five, more preferably at least six, still preferably at leastseven or at least eight orders-of-magnitude.

According to still another aspect of the present invention there isprovided a method of detecting binding between first member or membersof a binding pair and corresponding second member or members of thebinding pair. The method according to this aspect of the invention isimplemented by interacting a solid support onto which the first memberor members of the binding pair are immobilized and arrayed with thecorresponding second member or members of the binding pair. Thereafter,the spatial distribution of the second member or members of the bindingpair is determined at a sensitivity of detection which equals to or isgreater than 1 of 10 binding events, preferably, the sensitivity equalsto or greater than 1 of 5 binding events, more preferably, thesensitivity equals to about 1 of 1 binding events.

According to yet another aspect of the present invention there isprovided a method of detecting binding between first member or membersof a binding pair and corresponding second member or members of thebinding pair. The method according to this aspect of the invention isimplemented by interacting a solid support onto which the first memberor members of the binding pair are immobilized and arrayed with thecorresponding second member or members of the binding pair. Thereafter,the spatial distribution of the second member or members of the bindingpair at a signal-to-noise ratio greater than 20, preferably, greaterthan 50, more preferably, greater than 80, still preferably greater than100.

The first and second members of the binding pair according to thepresent invention can be of any biochemical or chemical nature and serveany physiological or therapeutic function. First and second members ofbinding pair according to the present invention, include, for example,antigen-antibody, antibody-antigen, hapten-antibody, antibody-hapten,nucleic acid-complementary nucleic acid, nucleic acid-substantiallycomplementary nucleic acid, ligand-receptor, receptor-ligand,enzyme-substrate, substrate-enzyme, enzyme-inhibitor andinhibitor-enzyme.

As used herein, the term “antigen” includes molecules having at leastone epitope recognized by an antibody. Such a molecule can be, forexample, a protein or a part thereof, a carbohydrate or a part thereofor any natural or man made chemical.

As used herein, the term “hapten” relates to a molecule or a portion ofa macromolecule to which an antibody may specifically bind.

As used herein, the term “antibody”, includes polyclonal antibody,monoclonal antibody, fragment of an antibody, single chain antibody anda chimeric antibody. The source of the antibody can be from the serum ofan immuned animal, a serum of a patient or produced by immortalizedcells, such as hybridomas or virus infected antibody producing cells.

As used herein, the term “nucleic acid” includes natural nucleic acidssuch as DNA and RNA, either derived from nature or syntheticallyprepared, as well as analog nucleic acids capable of base pairing withnatural nucleic acids.

As used herein, the phrase “complementary nucleic acid” refers to anucleic acid as this term is defined above having a sequence ofnucleobases, each of which matches a corresponding nucleobase in anothernucleic acid according to the base parity rules.

As used herein, the phrase “substantially complementary nucleic acid”refers to a nucleic acid having a sequence of nucleobases, most of which(e.g., above 50%, preferably above 60%, more preferably, above 70%,still preferably above 80%, yet preferably, above 90%) matchcorresponding nucleobases in another nucleic acid according to the baseparity rules.

As used herein the term “ligand” includes natural or man made moleculesor macromolecules which are capable of binding to a receptor. A ligandcan be, for example, a protein, a nucleic acid, a carbohydrate or asmall molecule, including for example lipids, steroids, etc. The ligandcan act as an agonist or antagonist when it binds the receptor. As such,a ligand can be a drug, a hormone, etc.

As used herein the term “receptor” includes macromolecules that bindligands. Such macromolecules may for example be proteinaceous, solubleor anchored to a membrane.

As used herein the term “enzyme” refers to a proteinaceous macromoleculehaving catalytic activity with respect to one or more substrates.

As used herein the term “substrates” refers to any kind of moleculewhich undergoes faster catalysis in the presence of an enzyme.

As used herein the term “inhibitor” includes any molecule capable ofreversibly or irreversibly slow down catalysis. As such, an inhibitorcan be a drug.

According to the present invention determining the spatial distributionof a heavy atom over the surface of the solid support is by particlescattering. Preferably, determining the spatial distribution of theheavy atom over the surface of the solid support is by electronscattering. Any and all devices capable of producing a particle beam andrecording scattered particles are suitable for implementing the presentinvention. Examples of such devices are described in more detailhereinafter.

The corresponding second member or members of the binding pair used inaccordance with the present invention can be either directly orindirectly tagged with a heavy atom. Preferably, the correspondingsecond member or members of the binding pair used in accordance with thepresent invention is indirectly tagged with a heavy atom. It will beappreciated by one of skills in the art that indirectly tagging thecorresponding second member or members of the binding pair provides anelement of universality to the method of the present invention. Theheavy atom can be any atom capable of scattering particles better thanthe atoms making organic molecules, such as C, H, O, N, S and P.Suitable heavy atoms include gold, silver and iron, which are frequentlyused in electron microscopy. Other heavy atoms, such as osmium andplatinum may also be considered. Methods are known to link such heavyatoms to a variety of organic molecules. For example, gold cancovalently bind through an SH group which is natural to proteins and canbe introduced in other molecules such as nucleic acids andpolysaccharides. Other heavy atoms can be trapped in suitable chelators,which can be linked to a variety of macromolecules using methods wellknown in the art.

According to an additional aspect of the present invention there isprovided a method of identifying and/or quantifying at least onebiological molecule in a sample. The method according to this aspect ofthe invention is implemented by contacting the sample with a microarraypresenting an addressable array of macromolecules of known identitiesunder conditions so as to allow binding between the at least onebiological molecule and the macromolecules of known identitiesThereafter, the spatial distribution of the at least one biologicalmolecule over a surface of the microarray is determined at a dynamicrange of linearity of at least four, preferably at least five, morepreferably at least six, still preferably at least seven or at leasteight orders-of-magnitude, thereby identifying and/or quantifying theleast one biological molecule in the sample.

According to still an additional aspect of the present invention thereis provided a method of identifying and/or quantifying at least onebiological molecule in a sample. The method according to this aspect ofthe invention is implemented by contacting the sample with a microarraypresenting an addressable array of macromolecules of known identitiesunder conditions so as to allow binding between the at least onebiological molecule and the macromolecules of known identities.Thereafter the spatial distribution of the at least one biologicalmolecule over a surface of the microarray is detected at a sensitivitywhich equals to or is greater than 1 of 10 binding events, preferably,equals to or is greater than 1 of 5 binding events, more preferably,equals to about 1 of 1 binding events, thereby identifying and/orquantifying the least one biological molecule in the sample.

According to yet an additional aspect of the present invention there isprovided a method of identifying and/or quantifying at least onebiological molecule in a sample. The method according to this aspect ofthe invention is implemented by contacting the sample with a microarraypresenting an addressable array of macromolecules of known identitiesunder conditions so as to allow binding between the at least onebiological molecule and the macromolecules of known identities.Thereafter the spatial distribution of the at least one biologicalmolecule over a surface of the microarray is detected at asignal-to-noise ratio greater than 20, preferably, greater than 50, morepreferably, greater than 80, still preferably greater than 100, therebyidentifying and/or quantifying the least one biological molecule in thesample.

According to still an additional aspect of the present invention thereis provided a method of identifying and/or quantifying at least onebiological molecule in a sample. The method according to this aspect ofthe invention is implemented by contacting the sample with a microarraypresenting an addressable array of macromolecules of known identitiesunder conditions so as to allow binding between the at least onebiological molecule and the macromolecules of known identities.Thereafter, the spatial distribution of the at least one biologicalmolecule over a surface of the microarray is detected by directly orindirectly tagging the at least one biological molecule with at leastone heavy atom and obtaining a particle scattering image of a spatialdistribution of the at least one heavy atom, thereby identifying and/orquantifying the least one biological molecule in the sample.

According to another aspect of the present invention there is provided amethod of identifying and/or quantifying at least one biologicalmolecule in a sample. The method according to this aspect of theinvention is implemented by attaching biological molecules present inthe sample to a solid support; contacting the solid support with atleast one macromolecule of a known identity under conditions so as toallow binding between the at least one biological molecule and the atleast one macromolecule of known identity. Thereafter, the level ofbinding between the at least one biological molecule and the at leastone macromolecule of known identity is determined at a dynamic range oflinearity of at least four, preferably at least five, more preferably atleast six, still preferably at least seven or at least eightorders-of-magnitude, thereby identifying and/or quantifying the leastone biological molecule in the sample.

According to still another aspect of the present invention there isprovided a method of identifying and/or quantifying at least onebiological molecule in a sample. The method according to this aspect ofthe invention is implemented by attaching biological molecules presentin the sample to a solid support; contacting the solid support with atleast one macromolecule of a known identity under conditions so as toallow binding between the at least one biological molecule and the atleast one macromolecule of known identity. Thereafter the level ofbinding between the at least one biological molecule and the at leastone macromolecule of known identity is detected at a sensitivity asensitivity which equals to or is greater than 1 of 10 binding events,preferably, equals to or is greater than 1 of 5 binding events, morepreferably, equals to about 1 of 1 binding events, thereby identifyingand/or quantifying the least one biological molecule in the sample.

According to a further aspect of the present invention there is provideda method of identifying and/or quantifying at least one biologicalmolecule in a sample. The method according to this aspect of theinvention is implemented by attaching biological molecules present inthe sample to a solid support; contacting the solid support with atleast one macromolecule of a known identity under conditions so as toallow binding between the at least one biological molecule and the atleast one macromolecule of known identity. Thereafter, the level ofbinding between the at least one biological molecule and the at leastone macromolecule of known identity is detected at a signal-to-noiseratio greater than 20, preferably, greater than 50, more preferably,greater than 80, still preferably greater than 100, thereby identifyingand/or quantifying the least one biological molecule in the sample.

According to still a further aspect of the present invention there isprovided a method of identifying and/or quantifying at least onebiological molecule in a sample. The method according to this aspect ofthe invention is implemented by attaching biological molecules presentin the sample to a solid support; contacting the solid support with atleast one macromolecule of a known identity under conditions so as toallow binding between the at least one biological molecule and the atleast one macromolecule of known identity. Thereafter the level ofbinding between the at least one biological molecule and the at leastone macromolecule of known identity is detected by directly orindirectly tagging the at least one macromolecule of known identity withat least one heavy atom and obtaining a particle scattering image of aspatial distribution of the at least one heavy atom, thereby identifyingand/or quantifying the least one biological molecule in the sample.

A method for the identification and quantification of biologicalmolecules on a preparate such as a microarray or a 2D-gel according topreferred embodiments of the present invention includes the followingsteps (i) localization (the biological molecules are separated inspace); (ii) tagging with markers (this step occurs before or after thelocalization, depending on the tagging, separation technology and/orpreparate); (iii) scanning the tags; (iv) identifying or quantifying thetags; (iv) interpreting the number of molecules in each location fromthe quantity of tags and relating the results to the biological problemthat is studied. According to the present invention, it is preferable toperform step (iii) with a scanning electron microscope (usingappropriate tags in step (ii)). This provides a detection method thatoperates at high sensitivity, broad dynamic range and low background.

In one embodiment of the invention, the method is carried out asillustrated schematically in the flow-chart of FIG. 1, and comprises thefollowing steps (1)-(9) (marked as 111-119 in FIG. 1):

-   -   1. Localizing the biomolecules in space.    -   2. Tagging the biomolecules with markers.    -   3. Preparing the preparate for vacuum.    -   4. Loading the preparate on X-Y-T stage (T means tilt) to        analysis position.    -   5. Scanning the preparate at low resolution to identify the        regions of interest (ROIs).    -   6. Scanning the ROIs with an electron beam at a high resolution.    -   7. Enhancing the image contrast.    -   8. Analyzing the ROIs.    -   9. Displaying results.

The step of localizing the biomolecules in space comprises any of thefollowing steps or combinations thereof:

-   -   A. Binding the biological molecules to a known or unknown        immobilized array of molecules.    -   B. Separating the biomolecules with one- or two-dimensional        electrophoresis (gels).    -   C. Attaching the biomolecules to a membrane (blot).    -   D. Observing the biomolecules' affiliation to a specific cell.    -   E. Separating the biomolecules by chromatography.    -   F. Separating the biomolecules in a flow system.

The step of tagging the biomolecules with markers can be done before orafter the above described stages, depending on the case. When thebiomolecules are DNA molecules, the tagging is preferably done beforethe spatial separation, while for proteins in the 2D-gel or microarraysthe tagging is preferably done after the spatial separation.

The tagging may comprises any combination of the following alternatives:

-   -   A. Use of heavy metal colloids (e.g., gold, silver), preferably        of a diameter range of 1-200 nm, whereby the colloids create a        high intensity back scattered electron signal and, therefore,        high image contrast.    -   B. Doping the biological molecule with a high atomic number        substance, to create a contrast sufficiently strong under an        electron beam. Preferably the doping substance is an organic        compound that contains iron.    -   C. A fluorescent electro-luminescent signal whereby the electron        beam creates a light signal that is detected. Such marking has        the advantage of high resolution electron beam inspection with        optical reading.

Tagging with more than one type of tags to make a distinction betweentwo preparates or different molecules is also possible. In the case ofcolloids, the tags may comprise different materials or of differentsizes. The material analysis can be done with EDS (Energy DispersiveSpectroscopy) yielding an X-ray spectrum. The differentiation on thebasis of size is done by image analysis.

For preparing the preparate for vacuum standard methods are used thatinclude:

-   -   A. Fixation e.g., with formaldehyde.    -   B. Coating with a conductive layer e.g., carbon.    -   C. Freezing to prevent out-gassing.

The preparation of the preparate for vacuum can be minimal when using anEnvironmental Scanning Electron Microscope (ESEM), as described furtherbelow.

After loading the preparate in the specimen chamber of an electronmicroscope, scanning of the preparate is carried out by reading at lowresolution either by the electron beam or with a combined opticalmicroscope, including optical fiber where access is indirect. In thisway, the regions of interest (ROIs) are identified and are then scannedat a higher or highest resolution.

The scanning of the ROIs with an electron beam at high resolution can bein one or two dimensions. The scanning in one dimension can besynchronous with the movement of the preparate in the second horizontaldimension.

To enhance the image contrast, algorithms well-known in the art can beused such as, for example, by calculating the histogram of pixel valuesand expanding it over the dynamic range of pixel values (typically2-255).

The analysis of the ROIs comprises any one of the following steps:

-   -   A. Performing edge detection algorithm to identify the colloids        in each ROI and counting said colloids.    -   B. Counting electro-luminescent signals.    -   C. Identifying X-ray spectrum of each particle for        identification by comparison to a reference spectrum.    -   D. Counting the signals by image analysis.

Display of the results may include presenting the number of molecules ineach ROI and the spatial map of the ROIs with indication of thedifferent types of molecules.

Reference is now made to FIG. 2 which is a general diagram showing alongitudinal cross-section of a SEM 1 used as a preparate analysisapparatus according to the present invention. Instruments similar to SEM1 are used for inspection of semiconductor wafers, as described, forexample, in U.S. Pat. Nos. 6,072,178, 5,644,132, 5,502,306, 4,618,938,4,609,809 and 4,618,938, the contents of which are herein incorporatedby reference as if fully disclosed herein.

A primary electron beam 12, travels through a vacuum path to reach apreparate 20. The electrons are emitted from an electron gun 3, poweredby a gun power supply which is electronically controlled as indicated by7. The beam is focused by a condenser lens 4 and an objective lens 5, toform a focal point on preparate 20. The beam is diverted by a deflector6 that scans the preparate in one or two dimensions. The preparate emitssecondary electrons (SE) 14, back scattered electrons (BSE) 16 andcharacteristic X-rays 8. The characteristic X-rays are subjected toenergy analysis to form and X-ray spectrum via an EDS. The BSE aredetected by the BSE detector 18. SE 14 are detected by the SE detector13. X-rays are detected by the EDS detector 150. The analog data isacquired and analyzed in a computer schematically represented by box101. Optical microscope assisted with optical fibers 104 is used for lowresolution inspection. A preparate that comprises biological moleculesis fed via a tray 102 into the SEM. The preparate compartment 103 isdivided to two sections in each of which pressurization can be performedindependently to permit loading or unloading of a preparate in onechamber (e.g., 103A) while simultaneously inspecting a second preparate(in e.g., 103B). Each of chambers 103A and 103B are large enough tocontain a 12″ (30 cm) diameter preparate 8. A stage 9 drives thepreparate under the scanning electron beam 12. The image is formed fromthe detected electron signal and is digitized for image processing.

Preparate 20 contains biological molecules that are separated in space.According to one embodiment of the present invention, preparate 20comprises a hybridized DNA microarray, as detailed further below.According to another embodiment of the present invention, the preparatecomprises a 2D PAGE as also detailed below. According to still anotherembodiment of the present invention, the preparate comprises a proteinmicroarray, a carbohydrate microarray or a cell microarray interactedwith any probe or target.

Reference is now made to FIG. 3, a generalized diagram showing across-section of a scanning electron microscope according to one aspectof the present invention. Parts that are the same as those shown in theprevious Figure are given the same reference numerals and are notdescribed again, except as necessary for an understanding of the presentembodiment.

According to this aspect of the present invention preparate 20 comprisesa DNA microarray 70, after hybridization. The substrate 72 is standard,e.g., glass, nitrocellulose membrane, nylon filter, filter paper, othersubstrates that are used in Southern, western or northern blots or inmicroarrays. The substrate can be silicon, glass, filter paper or anyother material that is convenient for microarray fabrication. Themiss-matched and perfect-match target-probes are marked by 26A and 26B,respectively. The technique of gold tags (discussed further below) isused.

In order to prevent charge accumulation, the microarray is coated by athin layer of a conductive material, schematically shown by the dashedline of 73. Preferably the coating material is carbon. Preferably thethickness range is 50 to 600 A. In order to make the preparate vacuumcompatible, it is fixed by standard electron microscopy methods,preferably by using formaldehyde. An alternative method to increase thecompatibility between the microarray 70 and the vacuum is by using acooling stage 74, preferably based on the Peltier effect. The stage isused to cool the microarray and thereby reduce the out-gassing.

The specific microarray preparate comprises domains of different probes26 as shown in FIG. 3 by way of a non-limiting example. A domain of basestructure TTGC 26A (SEQ ID NO:1), is shown as a representation of amiss-match. A domain of ATGC 26B (SEQ ID NO:2) represents perfectlymatched probes. The probes are hybridized with the target molecules 27.A plurality of tags 28 (e.g., gold colloids) is attached to the targetmolecules. In a preferred embodiment, there is one tag per target. Theadvantage of gold is that it creates a strong signal of back-scatteredelectrons due to its high atomic weight and corresponding highback-scattered electrons coefficient. This allows a high contrast, highresolution image at low current and exposure time, thus minimizing theradiation damage to the preparate.

It is possible to obtain commercial gold colloids as small as I nm (forexample, from Nanoprobes, Inc., 95 Horse Block Road, Yaphank, N.Y.11980-9710, USA). In order to get good correlation between the number ofgold particles and the number of hybridized DNA, it is important thatthe number of colloids per target will be constant, preferably one tagper probe. The binding between tags and targets should be stable at atemperature of 50° C., which is typical for hybridization. According toa yet another embodiment of the present invention, the tags areiron-rich molecules, as in the technology used by Clinical MicroSensors, Inc. (126 West Del Mar Blvd, Pasadena, Calif. 91105, USA). Theiron produces a sufficient contrast for the BSE signal.

The SEM/gold tags combination along with the ability to drive thepreparate mechanically enables quantitative microarray detection.According to the present invention, the colloids are counted from theacquired digital image. Then, the contrast of the image is enhancedusing a contrast enhancement algorithm. Then a pattern recognitionalgorithm identifies the colloids, for example by edge detection.Subsequently, the colloids are counted. This provides a quantitativemeasure of even the weakly expressed genes. A preferred reading strategythat reduces the reading time is to first scan the entire preparate andidentify the regions of interest and then rescan the regions of interestto the desired quality. The ability of SEM to work at resolutions thatvary from 10 microns down to about 1 nm, provides an ability to performsingle molecule detection on a very wide dynamic range.

The present invention is advantageous over existing methods since it isbased on single-molecule detection. This means that the sensitivity anddynamic range are considerably higher than in the presently usedfluorescence based methods. One advantage of single molecule detectionscheme is the fact that it is compatible with miniaturization of amicroarray. The miniaturization is desired since one would like to packcompactly as many molecules as possible on the same chip.

The following table summarizes a comparison of performance of the singlemolecule detection method of the present invention and the alternativefluorescence technology. A further advantage that relates to theminiaturization is the ability to use smaller preparates. In many casesonly a limited amount of sample is available. Exponential amplificationmethods such as PCR may alter the results in an uncontrolled manner. Theadvantages of a sensitive system that can detect smaller preparates areclear. TABLE 1 Fluorescence Electron Beam Detection Comparison:Detection Detection Resolution (pixel size) [unit] ˜10 μm <10 nm to ˜10μm Scan Time [min] ˜18 ˜ comparable (depends on number and size of ROIs)40 min to scan a whole microarray at a 100 nm resolution Sensitivity˜10⁵ 1 [molecules/100 μm²] (Number of molecules per micron squererequired for a signal) S/N ˜3-4 Much better, e.g., 20-100 Dynamic rangeof linearity ˜10³ ˜10⁸ Can rescan preparates N Y (no bleaching)Compatible with small N Y preparates (reducing PCR steps) Compatiblewith miniaturized N Y arrays

In another embodiment, the method of the present invention can becarried out at almost atmospheric pressure, for example using anEnvironmental Scanning Electron Microscope (ESEM), a commercial SEM thatworks at elevated pressures. Further information on ESEM and how itworks can be found in Enviromnental Scanning Electron Microscopy,Philips Electron Optics, Eindhoven, The Netherlands (Robert JohnsonAssoc. El Dorado Hills, Calif. 1996) as well as in U.S. Pat. Nos.5,250,808, 5,362,964 and 5,412,21 1, the contents of which are herebyincorporated by reference as if filly disclosed herein.

According to a yet another embodiment of the present invention, the ESEMis used for the inspection of the microarray of FIG. 2., without thepreparation for vacuum. In a preferred embodiment, the microarray willbe cooled by cooling plate 74, to reduce the vapor pressure. The mainadvantage of the ESEM is the ability to study topography in an elevatedpressure, utilizing a prior art secondary electrons (SE) detector. Theadvantage of the ESEM is that the hybridization can be detected simplyby measuring the density at each site. Thus, as mentioned below,according to one embodiment, the microarray will be analyzed withouttagging the targets. According to another embodiment of the presentinvention, the ESEM, or its specimen compartment, will be used insteadof the SEM disclosed in FIG. 2. The lower vacuum and the cooling relaxesthe steps needed to prepare the preparate for vacuum. For example, whenthe ESEM is used, it is possible to inspect 2D PAGEs and microarrayswithout fixation. An example of an apparatus that combined the ESEM withthe SEM disclosed above is given in FIG. 4. The SEM 60 contains apressure limiting aperture 63 that distinguishes between specimencompartment 103 and the column. In order to protect the back scatteredelectrons detector, it is enclosed in a protective chamber 61, the frontwindow of chamber 62, is a membrane transparent to electrons that canhold the pressure difference between the evacuated medium near thedetector and the gas. The secondary electrons are detected by the priorart ESEM SE detector.

According to a yet another embodiment of the present invention thetargets are not tagged at-all. The SEM is sufficiently sensitive todetect density differences between hybridized and non-hybridized regionsin nucleic acid microarrays and, similarly, interacted vs.non-interacted molecules in other types of microarrays, includingprotein and carbohydrate microarrays. A difficulty in using an electronbeam 12 for biomolecules is that it may damage the preparate. The damageto DNA, for example, from a beam of electrons is described in“Measurement of DNA damage by electrons with energies between 25 and4000 eV”, Folkard et al., Int J. Radiat. Biol. 64(6) pp 651-658 (1993).The choice of parameters should be safely below the damage. The use ofgold allows one to use small probe currents, in the range of 10 pA, andfast scanning, typically 10 or more frames per second, thus minimizingthe radiation absorbed by the DNA. In the areas where gold is present,it is expected that most of the interaction will be with the heavy goldatoms and not with the biomolecules. The main danger of radiation is theformation of free radicals in the water. Hence, according to a preferredembodiment of the present invention, a chemical that reduces theformation of free radicals but does not damage the biomolecules is addedto the microarray after hybridization/interaction and before inspection.Further information on these chemicals can be found in Siddiqi M. A. andBothe E., Radiation Research, Vol. 112, pp 449-463 (1987), the contentsof which are herein incorporated by reference.

Preferably, the microarray preparate is situated on a stage 21 that canbe moved by a servo motor 22. This arrangement drives the preparateunder the electron beam and effectively increases the scanned area. Themotor may incorporate an electrical vacuum feed-through 23. Such a motoris commercially available from Nanomotion, Ltd. (Mordot HaCarmelIndustrial Park, PO BOX 223, Yokneam, 20692, Israel). Alternatively, thestage can be moved by a conventional mechanical feed through. Accordingto yet another embodiment of the present invention motor 22 is situatedon an arm that drives it into the vacuum chamber via a load lock. Suchan arrangement improves the automation and elevates the throughput ofthe system. According to a preferred embodiment of the presentinvention, the preparate is driven along one axis (marked by X) and theelectrons beam scans the microarray along the perpendicular axis (markedby Y).

Due to the low signal-to-noise ratio inherent to microarrays, proteinmicroarrays in particular, as well as to other methods currently used todetect biological species, it is desirable to use comparative, ratherthan absolute measures. In the commonly used fluorescence-basedmicroarrays, this is done on the basis of different dye colors.According to a yet another embodiment of the present invention, this isdone in the SEM by means of an X-ray spectrum analysis. The X-ray isdetected by an EDS detector. The X-ray photons and the detector aremarked in FIG. 2 by 8 and 150, respectively. The characteristic X-rayspectrum of the tags serves for comparative study. As the colloids arecounted, their spectrum is acquired, analyzed and compared to areference spectrum. This method allows comparative study of more thanone tag since the number of possible X-ray spectrums is not limited.

Reference is now made to FIG. 5, that shows a yet another embodiment ofthe present invention, whereby the detection is made by exciting lightphotons (electro-luminescence). The target molecules are tagged withluminescent molecules 41, in a similar fashion to the prior artfluorescent tagging. According to the present invention, the luminescenttags 41 are excited by the electron beam 12 and/or the excited SE. Thelight beam 42 is guided to a photomultiplier (PMT), by means of a lightguide 43 (e.g., made of PMMA). The amplified light signal produced bythe PMT is transformed to an electrical signal at the SEM detector. Thedevice allows inspection of light emitted from the fluorescent DNAmolecules, at a resolution below 100 nm.

A further development that will increase throughput is parallelinspection with microcolumns. The microcolumns are miniature scanningelectron microscopes that are produced by integrated silicon processes.Due to their size, the microcolumns can operate in parallel,considerably reducing the scanning time and the bulkiness of a SEM basedsystem. Further details of the microcolumns are given in A. D. Feinermanand Crewe “Miniature Electron Optics”, Advances in Imaging and ElectronPhysics, Vol. 102, 187 (1998) as well as U.S. Pat. No. 5,122,663, thecontents of which are hereby incorporated by reference as if fullydisclosed herein.

Reference is now made to FIG. 6. According to the present invention, aplurality of microcolumns arranged in an array 80 is used for analysisof genes or proteins. Electrical wiring 81 controls the electron beamand lead the information from the detector to the data processingsystem. The beam of electrons scans the preparate of tagged nucleicacid, proteins, carbohydrates or cells in a microarray and/or 2D-gel, asappropriate. According to one embodiment of the present invention, thebiological preparate can be coated with a conducting material e.g.,carbon, as shown in 83. Alternatively, the preparate may be protected ina close chamber, as shown at 84 and the electrons will travel throughthe membrane.

According to an embodiment of the present invention, the SEM is used forthe analysis of proteins in a 2D PAGE. According to this embodiment,after counting, the preparate is prepared for mass spectrometry.According to this embodiment, the separation of proteins is done in a 2DPAGE. However, the present invention is compatible with other separationmethods, for example, electrophoresis in a fluid or through a membrane(e.g., as in a blot), chromatography (HPLC). The typical size of a 2DPAGE is 20×20 cm². This means that the entire 2D PAGE can easily fitinto the standard wafer compartment of a wafer inspection SEM (103 inFIG. 2.).

According to the present invention, the preferred tagging method is theattachment of gold colloids. This is done with known technologies suchas, for example, the one available from British Bio Cell Inc.(Cambridge, GB).

According to another aspect of the present invention tagging is done bysilver staining. Since there is no generic tagging that fits allproteins, the type of tagging to be used depends on the biologicalquestion that is asked. In many cases, the relevant question is whethera known protein exists in a preparate. In this case, the specifictagging of this protein, or number of proteins is applied and thedesired proteins can be read on a single molecule detection basis. Inother cases, general tagging, such as or silver staining can be applied.

According to the present invention, the preparation for vacuum is doneas follows: first the tagged molecules (proteins) are driven to thesurface by an electric field (shown schematically in FIG. 7 which isfurther referred to hereinbelow) and then the proteins are immobilizedon the surface. According to one embodiment of the present invention,the surface is made of silicon. According to another embodiment, thesurface is made of glass. After the proteins are attached to thesurface, the surface is detached from the gel, coated to prevent chargeaccumulation by the electron beam of the electron microscope and thenscanned thereby.

According to the present invention, the scanning is done by driving thepreparate mechanically under the electron beam in a continuous or a‘step and repeat’ manner. The driving is done in correlation withthe—scanning. According to one aspect of the present invention, thescanning is first done at a low resolution, to identify the ROIs, eitherautomatically via the software or manually. Then the preparate isscanned at a higher resolution to count the number of colloids in thesignificant spots.

Reference is now made to FIG. 7. The gel chamber 90 comprises 3 sets ofelectrodes. The electric field that separates the molecules is appliedby power supply V1 in the X direction and V3 in the Y direction. Theattachment to the upper surface is done via V2 (Z direction).

According to another embodiment of the present invention, the proteinsare marked with fluorescent or electro-luminescent molecules, similar tothe embodiment disclosed in FIG. 5 for DNA microarrays. According to oneembodiment of the present invention, the molecules are tagged beforethey are separated in the gel or attached to the microarray. Typicallyprotein analysis consists of two typical phases: separation orlocalization in space and identification via mass spectrometry,antibodies, etc. What is clearly missing is an intermediate stage wherethe number of proteins in each spot is counted. Preferably the countingmethod will be able to distinguish between different types of proteins.

According to the present invention there is disclosed a method ofprotein analysis that comprises of the following phases:

-   -   1. Localization via a 2D PAGE or on a microarray    -   2. Quantification (in an SEM)    -   3. Identification (preferably via mass spectrometry,        specifically Matrix Assisted Laser        Dissociation/Ionization-MALDI, antibodies).

In another embodiment of the present invention, proteins or proteinsamples, which can include proteins of a known identity or of an unknownidentity, naturally occurring or synthetic, antigens or antibodies,etc., are arrayed over a surface of a microarray and are immobilizedthereto and are thereafter interacted with appropriate directly orindirectly tagged macromolecules to generate a preparate suitable forelectron microscope inspection. Other preparate processing steps aresimilar to the steps described elsewhere herein.

In yet another embodiment of the present invention, saccharides orsaccharide samples, which can include saccharides of a known identity orof an unknown identity, naturally occurring or synthetic, are arrayedover a surface of a microarray and are immobilized thereto and arethereafter interacted with appropriate directly or indirectly taggedmacromolecules to generate a preparate suitable for electron microscopeinspection. Other preparate processing steps are similar to the stepsdescribed elsewhere herein.

In still another embodiment of the present invention, cells of a knownidentity or of an unknown identity are arrayed over a surface of amicroarray and are immobilized thereto and are thereafter interactedwith appropriate directly or indirectly tagged macromolecules togenerate a preparate suitable for electron microscope inspection. Otherpreparate processing steps are similar to the steps described elsewhereherein.

According to the present invention, the disclosed apparatus and methodcan be used for building databases. For example a database that aim atinterfacing protein information with DNA mapping and sequence data fromgenome projects. This may also include a file listing all of theinformation entered for the particular protein. An example of such adatabase, obtained by conventional means is described in: J. E. Celis,FEBS Letters 430, 64-72, 1998 which is incorporated herein by reference.

According to a preferred embodiment of the present invention awafer-inspection SEM is used to detect labeled molecules on microarrays.

Reference is now made to FIG. 8. The tagged proteins are immobilized ona surface 141. According to a preferred embodiment, the surface 141 iscoated with a thin layer of carbon 142. After immobilizing the proteins,the substrate is coated with an additional layer of carbon to preventcharge accumulation in the proteins. The substrate is then scanned inthe SEM.

Reference is now made to FIG. 9, showing gold conjugate proteins asimaged in the SEM. The image is shown ‘bare’ without contrastenhancement. It can be seen that an image analysis technique can beapplied to quantify the number of tags. This experiment has beenperformed with monoclonal antibody (mouse IgG1) 1E10 conjugated to 20 nmgold colloids. A silicon substrate was covered with a carbon layer of150-200 Angstrom. The substrate was attached to a conventional SEMaluminum support. On the silicon surface was a drop of antigen P277. Thedrop was dried in a vacuum oven at 40° C. for 20 minutes. The goldconjugated antibody was added by putting a drop on the silicon surface,in a way that covered all the surface. The antibody added was diluted1:10. The support was left in a humid chamber for 40 minutes. Theantibody was washed by dipping the support for a few seconds in PBS(phosphate-buffered saline) a few times and then in double-distilledwater.

As is discussed hereinabove and is further exemplified in the Examplessection that follows, the present invention teaches a method thatreaches single molecule detection levels, gives high signal-to-noiseratios, and demonstrates a very broad dynamic range, while retainingeasy preparate preparation. In its preferred embodiment, the method usesgold labeling and electron microscopy, preferably a Wafer-InspectionScanning Electron Microscope, to probe both protein function arrays andprotein detecting arrays and is demonstrated here using the sameimmobilization chemistry and robotics described by the prior art (G.MacBeath, Science 298 (2000) 1760-1763).

The reasons gold labeling and SEM scanning demonstrate such abilitiesare several and include:

-   -   1. Significant reduction of noise. Unlike fluorescence        techniques where auto fluorescence of the glass and the buffers        contribute to the noise, in the method of the present invention        false positive signals are only due to nonspecific binding of        the gold conjugated probe to the slide. This is because only        signal (back scattered electrons) coming from very heavy atoms        such as gold are detected by the SEM detectors.    -   2. In small enough dilutions, where the molecules attached to        the slide are in distances larger than the diameter of the gold        probe, only one gold particle can attach per probed protein or        ligand. This is because once a gold particle attached, it        occludes the molecule on the surface from other gold probes.        Since the SEM can detect single gold particles, this provides        single molecule detection capabilities.    -   3. Detection abilities are limited in the lower limit only by        false positive signals due to non-specific binding, and in the        upper limit by the highest number of colloids able to pack        closely in a given area in other words, the upper detection        limit can be controlled by the size of the gold colloids chosen        as probes. This ensures a very broad and highly linear dynamic        range. Also, detection abilities are not constrained by        instrumentation (unlike fluorescent methods where saturation of        the photodiodes in light detectors can occur).

In addition to the increased abilities in sensitivity and dynamic rangethere are several other advantages over other labeling methods such asfluorescence:

-   -   1. There is no bleaching of the signal. This means that the        preparate can be stored and scanned repeatedly across a long        period of time (e.g., in cases that inconsistencies were found        in the analysis or new data has accumulated that requires new        analysis of an old preparate).    -   2. The preparates have a higher reproducibility rate since there        is no dependence on the type of buffers and materials used, and        there is a weak dependence on the “Hands” preparing the        preparate.    -   3. In the case of gold tagged proteins, it seems the gold        enhances binding selectivity of protein bound to it, possibly        because in some cases there are several proteins per gold which        can increase the probability of binding. Another possibility is        that due to the large size of the gold only high affinity and        specific interactions survive the stringent washing conditions.

The potential of the method of the present invention for relatively highthroughput is demonstrated in the unrelated semiconductor industry whereSEMs are routinely used to scan wafers for microscopic defects andimpurities.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which describeexperiments aimed at testing the sensitivity (signal-to-noise ratio) andthe dynamic range of the method of the present invention. To this end,first the universal biotin-tagged avidin detection system was testedcomparatively to the prior art, to reveal that under variousexperimental conditions sensitivity is up to 100 fold improved, whereasthe dynamic range is both several orders-of-magnitude broader, with alower limit reaching the ultimate goal of single molecule detection.Then, the universal biotin-tagged avidin detection system was sandwichedto a hapten-antibody detection system and tested for its sensitivity anddynamic range.

Materials and Experimental Procedures

Arraying Proteins on Glass Slides:

Proteins were spotted on glass slides presenting aldehyde groups(Telechem International, SuperAldehyde Substrates) using a BioroboticsTAS arrayer. Three “flag” proteins (Biotin-BSA at a concentration of 0.1mg/ml) served as position pointers for each group of 4×4 spots. Spotteddrops were about 400 μm in diameter and 10 nano-liter (nl) in volume.256 protein spots were applied to each spotted area of a slide. Forfixation via the aldehyde groups, the spotted slides were incubated in ahumid chamber for 2-3 hours at ambient temperature. When required,spotted areas and/or subareas of a slide were spatially separated bysurrounding paraffin lines. In order to block free aldehyde groups, theslides were inverted and briefly placed in a solution of 1% BSA in PBS,pH 7.5, and then immediately immersed in a similar fresh solution for 1hour at room temperature with gentle agitation. Following a brief rinsein PBS, the slides were ready for further processing as described below.

Probing the Slides:

Gold conjugated streptavidin (gold-streptavidin) was purchased fromBritish Biocell International. Gold colloids were 20 nm (STP20) or 40 nm(STP40) in diameter. Gold-streptavidin was spun four times in a cooledcentrifuge at 12,000 revolutions-per-minute (RPM) for 20 minutes,resuspended twice in a fresh buffer containing 0.04% Tween20 (Sigma) and0.1% BSA (w/v); and twice in a fresh buffer containing 20% glycerol, 80%PBS, 0.1% BSA (w/v) and 0.5 M NaCl. Final concentration ofgold-streptavidin was approximately 15 nM for both STP20 and STP40.

Cy3-conjugated streptavidin was purchased from Amersham PharmaciaBiotech and diluted in a solution of 20% glycerol, 80% PBS, 0.1% BSA(w/v) to a concentration of 17 nM.

Slides were incubated for 4 hours with the gold-streptavidin, orCy3-streptavidin probes.

Preparing Gold Probed Slides for Electron Microscopy:

All gold-probed slides were immersed in a fixation/dehydration solution(3% paraformaldehyde and 2% glutaraldehyde) for 30 minutes, washed indouble distilled water (DDW) for an additional 10 minutes, spun for 5minutes at 1250 RPM to remove excess buffer, and dried in a vacuumchamber overnight. The slides were then cut into 1 inch² sections thatcontained all the spotted area, using a diamond glasscutter and attachedto electron microscope supports via a carbon double-sided tape. In orderto avoid charging of the samples, the glass sections were coated with a200 nm thin carbon coat using an Edward's carbon coater and their edgeswere colored with a conducting silver paste.

Scanning the Probed Slides:

Fluorescence of the Cy3-probed slides was scanned using a PackardScanArray 4000 scanner at a 20 μm resolution. Intensity was determinedby taking the average intensity of the pixels in corresponding spots inall slides and reducing the average intensity of the pixels immediatelysurrounding the corresponding spots. Typically, each data point wasbased on four individual experiments.

The gold-probed slides were visualized with a scanning electronmicroscope (SEM) (Jeol 6400).. Images of 48 μm² (for 40 nm goldcolloids) or 12 μm² (for 20 nm gold colloids, see FIG. 14) sized framesinside each spot were taken via a back scattered electrons detector.This detector can detect only electrons scattered from heavy atoms, andtherefore detects only the gold colloids and not any organic, lightweight atoms present. Gold colloids were counted using the NIH imageprocessing software (shareware downloadable from:http://www.pathsoc.org.uk/wwwboard/messages/214.html). Number of goldcolloids per spot, were taken to be the average number of gold colloidsin corresponding frames multiplied by the number of frames per spot(2.6103 of 48 μm² sized frames per spot in the case of 40 nm goldcolloids; 10.4-10³ of 12 μm² sized frames per spot in the case of 20 nmgold colloids).

BSA-Biotin—Gold/Cy3-Streptavidin Detection System:

BSA and biotin-caproate were purchased from Sigma. BSA-biotin conjugatewas prepared as follows: BSA (5 mg, 75 nmole) and biotin-caproate, (0.72mg, 1.9 μmole) were dissolved in ice cold 200 μl DMF and the mixture wasleft at room temperature for 2 hours. The number of biotin molecules perBSA molecule, estimating 50% conjugation efficacy is 12.5 on theaverage. Extensive dialysis was preformed against PBS to removeunconjugated biotin. Activity of the BSA-biotin was assayed employingELISA, using Horse Radish Peroxide conjugated to streptavidin as aprobe.

BSA-biotin, and BSA were dissolved in 40% glycerol, 60% PBS to aninitial concentration of 1 mg/ml. BSA-biotin was serially diluted 3-foldin 40% glycerol, 60% PBS, pH 7.5, 0.1% BSA (w/v). In all dilutions, thetotal amount of BSA (free BSA+biotinylated BSA) was kept constant at 1mg/ml. BSA-biotin was then spotted on the slides in differentconcentrations ranging from 1 mg/ml to 100 ng/ml. Free BSA, which servedas a control was also spotted on the slides. Slides were than processedas described above.

To probe the slides, 40 μl of gold-streptavidin (STP20 or STP40) orCy3-streptavidin were applied to each printed area and incubated for 4hours in a humid chamber at ambient temperature. Following incubation,the slides were washed 3 times, 3 minutes each time, with PBSsupplemented with 0.04% Tween20 (PBS/T). Cy3-streptavidin probed slideswere additionally rinsed twice in PBS, (3 minutes each rinse),centrifuged for 5 minutes at 1250 RPM to remove excess buffer, and leftto dry in a slide box. Gold-streptavidin probed slides were furtherprocessed for electron microscopy as described above. Slides werescanned as described above.

BSA-Hapten—Biotinylated Antibody—Gold Streptavidin Sandwiched DetectionSystem:

BSA-hapten 23.7 [1b (p-nitrobenzyl phosphonate N-glycylglutatarate)] wasprepared as described in Tawfik et al. Phosphorus and Sulfur, 1993, vol.76 123-126.

D2.3 antibody was prepared as described in Tawfik et al. Proc. Natl.Acad. Sci. USA, 1993, vol. 90 p. 373-377.

The affinity constant for hapten 23.7 and D2.3 antibody in solution wasdetermined to be 4 nM by competitive ELISA (Tawfik et al. (1997) Eur. J.Biochem. vol. 244 p. 619-626) and further by a fluorescence assay(Lindner et al. (1999) J. Mol. Biol. vol. 285 p. 421-430).

Biotin-caproate was purchased from Sigma. 90 μl of biotin-caproate (20μmole) were dissolved in a solution having a total volume of 1 ml andcontaining 336 μl D2.3 antibody (1 mg, 6.7 nmole) in PBS and NaHCO₃ (1M; 100 μl). The reaction mixture was placed on ice for 3 hours, followedby extensive dialysis against PBS. Activity of the D2.3 antibody wasassayed with ELISA SA-HRP/GaM HRP.

BSA-hapten 23.7 was dissolved in 40% glycerol, 60% PBS, pH 7.5 at aconcentration of 200 μg/ml and spotted on slides as described above. Theslides were then further processed as described above.

The control, non biotinylated D2.3 (5 ng/ml), was dissolved in 20%glycerol, 80% PBS, 0.1% BSA (w/v). The biotinylated D2.3 antibody wasdiluted in a solution containing 20% glycerol, 80% PBS and 0.1% BSA(w/v). Dilutions ranged from 50 μg/ml biotinylated D2.3 antibody to 0.5ng/ml biotinylated D2.3 antibody. Twenty μl of each dilution and controlwere applied to separate sections of the slides. Following 2-3 hourincubation in a humid chamber at ambient temperature, the slides werewashed 3 timed (3 minutes each wash) with PBS/T.

To probe the slides, 40 μl of gold-streptavidin were applied to eachprinted area and incubated for 4 hours in a humid chamber at ambienttemperature. Following incubation, the slides were washed 3 times for 3minutes each time in PBS/T. Gold-streptavidin probed slides were furtherprocessed for electron microscopy as described above. All slides werescanned as described above.

Experimental Results

BSA-Biotin—Gold/Cy3-Streplavidin Universal Detection Systems:

The results for the BSA-biotin—gold/Cy3-streptavidin universal detectionsystem are shown in FIGS. 10-14, clearly demonstrating the far superiorsensitivity (higher S/N ratios) and dynamic range of the presentinvention over the prior art in any and all of the experimentalconditions employed. A few interesting points arise from the results.The first is that gold probes (STP20) extend the dynamic range (i.e.,the range where signal scales linearly with protein concentration) andthe sensitivity of detection by almost 100-fold relative to fluorescenceprobes (see, FIGS. 10-12). Another result is that 20 nm gold colloidsperformed far better than 40 nm gold colloids (improving signal-to-noiseby about 4-fold, or [R40/R20]2, see FIG. 10 and 11). The latter resultindicates that using even smaller gold colloids, e.g., 10 nm and 5 nmcolloids, will improve sensitivity and extend the lower limit of thedynamic range by an additional 10-100 fold relative to the fluorescenceprobe. Since the largest number of colloids able to pack closely in agiven area limits the upper detection limit, it is expected thatdecreasing the size of the colloids from 20 nm to 5 nm will furtherextend the dynamic range in its upper limit by a factor of at least 16fold. Moreover, by careful normalization of measured results, it will bepossible to carry out relative measurements of protein samples on thesame microarray using distinguishable sizes of colloids.

In order to approximate the detection abilities of the system, the ratiobetween the approximated number of BSA-biotin molecules conjugated tothe glass surface in one spot, and the number of gold colloids detectedin a spot was evaluated. To estimate the number of biotin labeled BSAmolecules conjugated to the glass in each spot, first the number ofaldehyde groups per spot was calculated by multiplying the number ofaldehyde groups per cm² (5·10¹² groups per cm²) by the area covered by adrop of a 400 μm in diameter (1.25·10⁻³ cm²). The result is 6·10⁹aldehyde groups per spot. The number of BSA molecules contained in a 10nl droplet of a 1 mg/ml BSA solution is 9·10¹⁰ molecules, which meansthat a maximal attachment of <10% of the protein molecules in a dropletcan be achieved. To estimate the number of gold colloids detected in aspot, the average number of gold colloids counted per frame wasmultiplied by the number of frames contained in the area covered by thedrop (see above).

FIG. 13 presents the estimated numbers, assuming a maximal attachment of10%. Taking the slope of the linear fit, and taking into considerationthat the aldehyde quantification was not done by protein attachment butrather by attachment of small molecules, the real value of proteinattachment is probably <10%, hence, the detection is close to 1:1 of allmolecules present in a spot. In other words, assuming that 10% of thebiotin molecules floating in the spotted drop also conjugatesuccessfully to the glass surface via the aldehyde groups thereat, thedetection is at worst 1 of every 4, but more likely closer to detectingall biotin molecules. This experiment demonstrates that by using themethod of the present invention, the lower possible limit of the dynamicrange, i.e., every single molecule detection, was reached or nearlyreached.

BSA-Hapten—Biolinylated Antibody—Gold Streptavidin Sandwiched DetectionSystem:

To determine the sensitivity to concentration and as a demonstrativeapplication for the method of the present invention a model system basedon a sandwich detection system—BSA-hapten—biotinylated antibody—gold/Cy3streptavidin—was employed. In the model system, a hapten conjugated toBSA was spotted on glass slides. It was then interacted with differentconcentrations of a corresponding biotinylated monoclonal antibody. Thecomplex BSA-hapten-antibody-biotin was thereafter probed with STP40.

The results which are shown in FIG. 15 reveal that even atconcentrations as low as a few tens of picomolars of antibody, detectionwas successful.

In order to obtain another approximation of the detection abilities ofthe method of the present invention, equilibrium equations were used tocalculate how many hapten-antibody complexes are expected to form underthe experimental conditions employed. The affinity constant (K=4 nM) wasassumed to be the same as when both hapten and antibody are free insolution, based on 2 independent measurements conducted in Fluorescenceand ELISA assays (see methods). The efficiency of attachment of theBSA-hapten to the substrate, taking into account the binding capacity ofthe aldehyde groups was assumed to be <30%. Then the ratio of the numberof complexes detected by the gold colloids to the maximal number ofcomplexes expected to exist on the glass was calculated. A ratio closeto 1:10 was obtained, indicating that method detects about one of everyten complexes that are actually formed on the slide. In view of thecalculations above, it is again anticipated that using smaller goldcolloids this ratio will substantially improve.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

1-105. (canceled)
 106. A method for detecting binding between all leastone first member of a binding pair and at least one corresponding secondmember of the binding pair, the method comprising: interacting a solidsupport auto which said at least one first member of the binding pair isimmobilized and arrayed with said at least one corresponding secondmember of the binding pair, wherein said at least one correspondingsecond member of the binding pair is at least one of: (a) directlytagged; and (b) indirectly tagged; with a heavy atom; and determining aspatial distribution of said heavy atom over a surface of the solidsupport so as to detect binding between said al least one first memberof the binding pair and said at least one corresponding second member ofthe binding pair.
 107. The method of claim 106, wherein said determiningthe spatial distribution of said heavy atom over said surface of thesolid support has a dynamic range of linearity of at least fourorders-of-magnitude.
 108. The method of claim 106, wherein saiddetermining the spatial distribution of said heavy atom over saidsurface of the solid support is at a sensitivity of detection equal toat least 1 in 10 binding events.
 109. The method of claim 108, whereinsaid sensitivity is equal to at least 1 in 5 binding events.
 110. Themethod of claim 109, wherein said sensitivity is about 1 in 1 bindingevents.
 111. The method of claim 106, wherein said determining thespatial distribution of said heavy atom over said surface of the solidsupport is at a signal-to-noise ratio of more than
 20. 112. The methodof claim 106, wherein said determining the spatial distribution of saidheavy atom over said surface of the solid support is at asignal-to-noise ratio of more than
 50. 113. The method of claim 106,wherein said determining the spatial distribution of said heavy atomover said surface of the solid support is at a signal-to-noise ratio ofmore than
 80. 114. The method of claim 106, wherein said binding pair isselected from the group consisting of antigen-antibody,antibody-antigen, hapten-antibody, antibody-hapten, nucleicacid-complementary nucleic acid, nucleic acid-substantiallycomplementary nucleic acid, ligand-receptor, receptor-ligand,enzyme-substrate, substrate-enzyme, enzyme-inhibitor andinhibitor-enzyme.
 115. The method of claim 106, wherein said determiningthe spatial distribution for said heavy atom over said surface of thesolid support is by particle scattering.
 116. The method of claim 106,wherein said determining the spatial distribution for said heavy atomover said surface of the solid support is by electron scattering. 117.The method of claim 106, wherein said heavy atom is selected from thegroup consisting of gold, silver and iron.
 118. A method for detectingbinding between at least one first member of a binding pair and at leastone corresponding second member of the binding pair, the methodcomprising: interacting a solid support onto which said at least onefirst member of the binding pair is immobilized and arrayed with said atleast one corresponding second member of the binding pair; anddetermining a spatial distribution of said at least one correspondingsecond member of the binding hair at a signal-to-noise ratio of morethan
 10. 119. The method of claim 118, wherein said at least one firstmember comprises at least one biological molecule is selected from thegroup consisting of a protein, a glycoprotein, a nucleic acid and acarbohydrate.
 120. The method of claim 118, wherein said at least onesecond member comprises at least one macromolecule of a known identityselected from the group consisting of proteins, glycoproteins, nucleicacids and carbohydrates.
 121. A method for at least one of identifyingand quantifying at least one biological molecule in a sample, the methodcomprising; contacting the sample with a microarray presenting anaddressable array of macromolecules of known identities under conditionsso as to allow binding between said at least one biological molecule andsaid macromolecules of known identities; and detecting a spatialdistribution of said at least one biological molecule over a surface ofsaid microarray at a sensitivity equal to at least 1 in 10 bindingevents so as to provide at least one of an identification and aquantification of the least one biological molecule in the sample. 122.The method of claim 121, wherein said at least one biological moleculeis selected from the group consisting of a protein, a glycoprotein, anucleic acid and a carbohydrate.
 123. The method of claim 121, whereinsaid macromolecules of known identities are selected from the groupconsisting of proteins, glycoproteins, nucleic acids and carbohydrates.124. The method of claim 121, wherein said detecting the spatialdistribution of said at least one biological molecule over said surfaceof said microarray has a dynamic range of linearity of at least fourorders-of-magnitude.
 125. The method of claim 121, wherein saiddetecting the spatial distribution of said at least one biologicalmolecule over said surface of said microarray has a signal-to-noiseratio of more than
 20. 126. The method of claim 121, wherein saiddetecting the spatial distribution of said at least one biologicalmolecule over said surface of said microarray is by directly orindirectly tagging said at least one biological molecule with at leastone heavy atom and obtaining a particle scattering image of a spatialdistribution of said at least one heavy atom.
 127. The method of claim121, wherein the signal-to-noise ratio is greater thin
 20. 128. A methodfor at least one of identifying and quantifying at least one biologicalmolecule in a sample, the method comprising: attaching biologicalmolecules present in the sample to a solid support; contacting saidsolid support with at least one macromolecule of a known identity underconditions so as to allow binding between said at least one biologicalmolecule and said at least one macromolecule of known identity; anddetecting a level of binding between said at least one biologicalmolecule and said at least one macromolecule of known identity bydirectly or indirectly tagging said at least one macromolecule of knownidentity with at least one heavy atom and obtaining a particlescattering image of a spatial distribution of said at least one heavyatom, thereby providing at least one of an identification and aquantification of the least one biological molecule in the sample. 129.The method of claim 128, wherein said at least one biological moleculeis selected from the group consisting of a protein, a glycoprotein, anucleic acid and a carbohydrate.
 130. The method of claim 128, whereinsaid at least one macromolecule of known identity is selected from thegroup consisting of a protein, a glycoprotein, a nucleic acid and acarbohydrate.
 131. The method of claim 128, wherein said detecting thelevel of binding between said at least one biological molecule and saidat least one macromolecule of known identity has a dynamic range oflinearity of at least four orders-of-magnitude.
 132. The method of claim128, wherein said detecting the level of binding between said at leastone biological molecule and said at least one macromolecule of knownidentity is at a sensitivity equal to at least 1 in 10 binding events.133. The method of claim 128, wherein said detecting the level ofbinding between said at least one biological molecule and said at leastone macromolecule of known identity is at a signal-to-noise ratio ofmore than
 20. 134. A method for at least one of identifying andquantifying biological molecules in a preparation, the methodcomprising: localizing and tagging the biological molecules in thepreparation; preparing the preparation for vacuum; loading thepreparation into the specimen chamber of an electron beam device;irradiating the preparation with an electron beam, thus obtaining animage of the tags; and analyzing the image of the biological moleculesby image analysis software so as to provide at least one of anidentification and a quantification of the biological molecules in saidpreparation.
 135. A method for at least one of identifying andquantifying biological molecule in a preparation, the method comprising:localizing and tagging the biological molecules in the preparation;lading the preparation into the specimen clamber of an electron beamdevice; irradiating the preparation with an electron beam, thusobtaining an image of the tags; and analyzing the image of thebiological molecules by image analysis software so as to provide atleast one of an identification and a quantification of the biologicalmolecules in sail preparation.
 136. An apparatus for analysis of atleast two samples comprising biological molecules, comprising: anelectron source to provide an electron beam; a charged particle beamcolumn to deliver and scan an electron beam from said electron source onthe surface of a first sample of said at least two samples; a vacuumsystem including a first and a second chamber in each of whichpressurization can be performed independently to permit loading orunloading of a second sample of said at least two samples in one chamberwhile simultaneously inspecting said first sample; at least one electrondetector; a measuring system for measuring X-ray spectrum; acontinuously moving x-y stage disposed to receive said second sample andto provide at least one degree of motion to said second sample while thefirst sample is being scanned; and an image analysis system forcarrying, out image analysis of the molecules of the first and secondsamples.